Age-related retinal degeneration resulting from the deletion of Shp2 tyrosine phosphatase in photoreceptor neurons

Shp2, a critical SH2-domain-containing tyrosine phosphatase, is essential for cellular regulation and implicated in metabolic disruptions, obesity, diabetes, Noonan syndrome, LEOPARD syndrome, and cancers. This study focuses on Shp2 in rod photoreceptor cells, revealing its enrichment, particularly in rods. Deletion of Shp2 in rods leads to age-dependent photoreceptor degeneration. Shp2 targets occludin (OCLN), a tight junction protein, and its deletion reduces OCLN expression in the retina and retinal pigment epithelium (RPE). The isolation of actively translating mRNAs from rods lacking Shp2, followed by RNA sequencing, reveals alterations in cell cycle regulation. Additionally, altered retinal metabolism is observed in retinal cells lacking Shp2. Our studies indicate that Shp2 is crucial for maintaining the structure and function of photoreceptors.


INTRODUCTION
Src homology region 2 (SH2)-containing protein tyrosine phosphatase 2 (Shp2), is a non-receptor phosphotyrosine phosphatase, encoded by the ptpn11gene [1,2].Shp2 is a cytoplasmic phosphatase ubiquitously expressed in cells and various tissues [2].Shp2 regulates both receptor tyrosine kinase and non-receptor tyrosine kinaseregulated pathways [2][3][4].Shp2 activity plays an important role in regulating tumorigenesis, development, and metabolic diseases [5,6].An earlier study shows that Shp2 protein expression is absent from the photoreceptor cells and loss of Shp2 in the retina results in photoreceptor degeneration and suggests that Shp2 may be indirectly required for photoreceptor cell survival, a non-cellautonomous function of Shp2 [7].Interestingly, we previously reported that the Shp2 substrate, Grb2-associated binding protein 1 (Gab1) is predominantly expressed in photoreceptor cells [8].These observations raise several questions, which include 1) whether Shp2 is expressed in photoreceptor cells, and 2) how photoreceptor degeneration occurs in the retina-specific deletion of Shp2, which we addressed in this study.We have employed state-of-the-art biochemical, bioinformatics, and functional techniques to demonstrate that Shp2 is expressed in photoreceptors and rod-cell-specific deletion of Shp2 results in photoreceptor degeneration.Our studies suggest that Shp2 is essential for the maintenance of the structure, function, and viability of photoreceptor cells.

Characterization of Shp2 in rod photoreceptors
To determine whether Shp2 is expressed in rod photoreceptor cells, we have isolated bovine rods, which contain both rod outer and inner segments co-immunolabelled with rhodopsin and Shp2 antibodies.We found the expression of Shp2 in both the rod outer and inner segments whereas the rod outer segment marker, rhodopsin exclusively stained the outer segments (Fig. 1A-C).To validate further the expression of Shp2 in rod photoreceptors, we isolated intact photoreceptors on Opti-prep density gradient centrifugation, and the fractions were immunoblotted with Shp2 and rhodopsin antibodies.Our results further show that Shp2 coeluted with rhodopsin suggesting the expression of Shp2 in rod photoreceptor cells (Fig. 1D).
Retina is a heterogeneous tissue composed of several cell types (Fig. 1E).To further examine the cell-type specific expression of Shp2, we employed Translating Ribosome Affinity Purification (TRAP) by breeding RiboTag mice which allows for Cre-mediated labeling of ribosomes with an HA-tagged ribosomal protein Rpl22; with mice carrying a rod specific Cre, allowing for the labeling of Rod Polysomes with HA.Immunoprecipitation of polysomes with antibody against HA results in the isolation of actively translating mRNA and quantitative real-time PCR (qRT-PCR) with gene-specific primers reveal the quantitative expression of the gene of interest (Fig. 1F).In this study, we bred RiboTag mice with rod photoreceptor-specific rhodopsin-Cre, cone photoreceptorspecific red/green opsin-Cre, retinal pigment epithelial-specific VMD2-Cre, Müller cell-specific Rax-Cre and AAV2-CMV-Cre to target retinal ganglion cells as we described earlier [9].Isolated polysome-bound actively translating mRNA was subjected to qRT-PCR with Shp2 primers (Table 1) and normalized the expression to Rpl37 ribosomal protein [9].Our results indicate increased expression of Shp2 in rods followed by cone, Müller cells, and RPE compared to input (total RNA isolated from respective Cre/ Rpl22 mouse retina) and ganglion cells (Fig. 1G).We previously reported that Shp2 substrate Gab1 is predominantly expressed in rod inner segments [8].Our TRAP data indicate increased expression of Gab1 in both rods and cones but not in RPE, Müller and ganglion cells (Fig. 1G).Collectively, our data confirm the expression of Shp2 in rod photoreceptor cells.

Functional role of Shp2 in rod photoreceptor cells
We generated rod-specific Shp2 KO mice under the control of rhodopsin promoter [10] (Fig. 2A).Quantitative real-time PCR analysis on Shp2 KO ( rod Shp2 −/− ) mice show a significant decrease of Shp2 expression compared to wild type (Shp2 flox/flox ) mice (Fig. 2B).There is a lack of Shp2 antibodies which are suitable for immunofluorescence, we immunoblotted the Shp2 flox/flox and rod Shp2 −/− mouse retinas with a Shp2 antibody showing a 60% decrease of Shp2 levels in rod Shp2 −/− mice compared to Shp2 flox/ flox mouse retinas, consistent with the Cre-antibody reactivity only in rod Shp2 −/− mouse retina (Fig. 2C, D).In rod Shp2 −/− mouse retina, the remaining 40% of Shp2 protein likely comes from other retinal cell types.At 6 weeks of age, there was no significant difference in rod scotopic a-(Fig.2E) and b-wave (Fig. 2F) and cone photopic b-wave (Fig. 2G) amplitudes between rod Shp2 −/− and Shp2 flox/flox mice.Consistent with the function, there was no significant difference in the morphology of the retina between these two genotypes (Fig. 2H).At 20 weeks of age, rod Shp2 −/− mice showed a significant reduction in scotopic a-wave (Fig. 2I) and b-wave (Fig. 2J) amplitudes compared to Shp2 flox/flox mice.However, there was no significant difference in photopic b-wave amplitudes between Shp2 flox/flox and rod Shp2 −/− mice (Fig. 2K).At this time point, we found thinning of both rod outer segment (ROS) and outer nuclear layer (ONL) thickness in rod Shp2 −/− mice compared to Shp2 flox/flox mice (Fig. 2L).At 64 weeks, rod Shp2 −/− mice exhibited a complete loss of scotopic a-wave (Fig. 2M), significantly reduced scotopic b-wave (Fig. 2N) and photopic b-wave amplitudes compared to Fig. 1 Expression of Shp2 in various retinal cells.Isolated bovine rods were immunostained with Shp2 (green) and rhodopsin (red) antibodies (A-C).ROS, rod outer segments, RIS, rod inner segments, CC, connecting cilium.Retina lysate was subjected to Opti-Prep (8-40%) density gradient centrifugation and the fractions were immunoblotted with Shp2 and opsin (rhodopsin) (D).Cross section of the retina displaying all the retinal cells (E).Translating Ribosome Affinity Purification.Floxed Rpl22 mice were mated with rod-Cre (F), cone-Cre, Müller cell-Cre, RPE-Cre, and RGC-Cre individually and the ribosomes were immuno-affinity purified with HA antibody.The bound mRNA associated with ribosomes was isolated (F).Equal amounts of mRNA from various retinal cells were quantified using Shp2 and Gab1 primers, and the data was normalized to the housekeeping gene Rpl37 (G).Input represents the total retinal RNA.Data are mean ± SEM (n = 3).Panels (E, F) were created with BioRender.com.Note: The opsin blot shown in (D) was previously used in another study [41].The same samples were used to examine Shp2 expression.
Shp2 flox/flox mice.Morphological analysis revealed the loss of ROS and ONL layers (Fig. 2P), indicating that Shp2 deficiency in rods leads to age-related photoreceptor degeneration.To confirm the age-related morphological changes in rod Shp2 −/− mice, we performed immunoblotting of retinal proteins from Shp2 flox/flox and rod Shp2 −/− mice using a rhodopsin antibody.The results showed no change in rhodopsin levels between Shp2 flox/flox and rod Shp2 −/− mice at 6 weeks of age (Fig. 2Q, T).However, there was a significant decrease in rhodopsin levels at 20 weeks (Fig. 2R, T) and a complete absence of rhodopsin at 64 weeks (Fig. 2S, T) in rod Shp2 −/− mice compared to Shp2 flox/flox mice.These findings further confirm that the loss of Shp2 in rods leads to age-related photoreceptor degeneration.

Biochemical studies on Shp2 KO mice
Recent studies show that Occludin (OCLN) is a Shp2 substrate [11].OCLN is a novel integral membrane protein localizing at tight junctions [12].Furthermore, tight junction protein interactions and barrier function is mediated through OCLN S408 phosphorylation [13].OCLN is also expressed in cells without tight junctions suggesting additional functions that are unrelated to tight junction formation [14].We examined OCLN levels in TRAP samples isolated from retinal cell types show the highest expression in retinal pigment epithelium (RPE) followed by rods and cones whereas Müller cells do not express OCLN (Fig. 3A).These studies show that OCLN is not only expressed in RPE as it is required for tight junction formation but also expressed in both rod and cone photoreceptor cells.
To confirm that OCLN is expressed in the retina, we prepared retina and RPE and the protein lysates were immunoblotted with OCLN, rhodopsin, and RPE65 antibodies.The results show that rhodopsin is expressed in the retina but not in the RPE, whereas RPE65 is expressed in the RPE but not in the retina (Fig. 3B).Under these conditions, we found the expression of OCLN in both the retina and RPE (Fig. 3B), suggesting that OCLN is present in both tissues.To examine whether OCLN is expressed in rod photoreceptor cells, we isolated these cells using discontinuous sucrose density gradient centrifugation.This process yields pure outer segment membranes (ROS) and an enriched inner segment fraction (RIS), which also includes other inner retinal cells [9].These fractions were immunoblotted with OCLN and rhodopsin antibodies, and the results demonstrate that increased OCLN is associated with RIS fractions compared to ROS; nevertheless, ROS also shows the expression of OCLN (Fig. 3C).To determine whether the loss of Shp2 in rod photoreceptor cells affects OCLN levels in the retina, we examined OCLN levels in Shp2 flox/flox and rod Shp2 −/− mouse retinas.The results show significantly reduced OCLN levels in rod Shp2 −/− retinas compared to Shp2 flox/flox retinas (Fig. 3D, E).This suggests that Shp2 plays a role in the expression or turnover of OCLN in photoreceptor cells.
Our TRAP data show increased expression of OCLN in the RPE (Fig. 3A).To examine whether the loss of Shp2 in rods affects OCLN expression in the RPE, we prepared RPE flat mounts from five-month-old Shp2 flox/flox and rod Shp2 −/− mice and stained them with OCLN and Phalloidin.The results revealed intense OCLN staining in the RPE of Shp2 flox/flox , whereas there was a complete loss of OCLN staining in the RPE of rod Shp2 −/− mice (Fig. 3F, G).These findings suggest that the loss of Shp2 in rods affects the expression or turnover of OCLN in the RPE.

Interaction of Shp2 with OCLN in a heterologous system
Recent studies show that OCLN is a Shp2 substrate [11].Mutation of Asp to Ala in the conserved WPD loop in the protein tyrosine phosphatases (PTPs) acts as a high-affinity trap to lock their substrates [15].Several studies have identified PTP substrates using this technology [8,15].Shp2 "Substrate-trapping" mutants have been used to identify Shp2 substrates [16].We generated three Shp2 constructs (Shp2-WT, Shp2-D425A, and Shp2-D425A/ C459S) with a HA-tagged epitope.Flag-tagged OCLN and HAtagged WT and mutant Shp2 constructs were either expressed alone (Fig. 3H) or co-expressed (Fig. 3I) in HEK-293T cells.Coexpression of HA-Shp2-D425A mutant with Flag-OCLN resulted in proteolysis (Fig. 3I).Co-immunoprecipitation experiments (Fig. 3J-M) suggest that Flag-OCLN can associate with HA-Shp2-WT and HA-Shp2-D425A/C459S and vice versa (Fig. 3J, K).These observations suggest that OCLN interacts with Shp2 in vitro.We could not identify the in vivo interaction between Shp2 and OCLN, as we do not have good antibodies to study their physical interaction via immunofluorescence or co-immunoprecipitation.Table 1.Real-time PCR primers to quantitate the expression of Shp2, Gab1, Occludin, and inflammatory markers.

Gene
Forward Primer Reverse Primer

Shp2-regulated gene expression in rod photoreceptor cells
We generated Rod-specific Shp2/ Ribo-Tag mice by first breeding homozygous Shp2 floxed mice with homozygous Ribo-Tag mice.Simultaneously, we bred homozygous Ribo-Tag mice with rhodopsin-Cre mice.Males who carried one allele of Shp2, were homozygous for Ribo-Tag, and carrying the rhodopsin Cre was then mated to females who were homozygous for both Shp2 and Ribo-Tag (Fig. 4A).As a result, Cre-positive mice who were heterozygous for ( rod Shp2 +/-: Control) were compared to Crepositive homozygous mice ( rod Shp2 −/− : KO); additionally, all experimental mice were homozygous for Ribo Tag.Polyribosome immunoprecipitation was then carried out on retinas, as described previously [9] and both input and HA-IP fractions were subjected to RNA sequencing at MedGenome Inc (Foster City, CA).RNA sequencing resulted in reads on ~14,417 genes in our retina samples and ~11,600 genes in our rod-enriched samples.GO Analysis (http://geneontology.org/) of the Top 500 enriched genes after Rod-TRAP showed transcripts involved in processes of visual perception, photoreceptor function and development, and ciliary assembly.Further validating Rod-TRAP as a suitable method to enrich Rod Specific transcripts (Fig. 4B).Rod-TRAP transcripts were also depleted for non-Rod photoreceptor markers, as well as mitochondrial genes (Fig. 4D, E).This observation was further confirmed with unsupervised hierarchical clustering of Rod-TRAP and retinal samples, showing clustering between Rod-TRAP and retinal samples, and subclustering of Shp2 WT and Shp2 null Rod-Trap Samples.(Fig. 4C) A comparison between retinal mRNA samples of identified three differentially regulated genes Slc29a1, Rps18-ps5, and Gm49909.(Fig. 4F, G).To identify alterations between rod-enriched mRNA samples of rod Shp2 +/-and rod Shp2 −/− , we removed all rod-depleted genes from our rod translatome datasets; this was done to ensure we assayed for changes occurring only with rod-specific transcripts.A comparison between rod-enriched mRNA samples of rod Shp2 +/-and rod Shp2 −/− identified 2581 differentially regulated genes between samples (Fig. 4H, I); these genes were then used for Ingenuity Pathway Analysis (IPA).
Unsupervised hierarchical clustering was also performed on the translatomes between the Rod Fractions of rod Shp2 WT and KO mice; using metabolic gene panels designed for Glycolysis, Pentose Phosphate Shunt (PPS), TCA Cycle, Mitochondria, Fatty Acid Oxidation (FAO) and Antioxidants (Fig. 5).The separation between rod Shp2 WT and KO mice could be observed in Glycolysis, PPS, TCA, and Mitochondria, but not in FAO or Antioxidant Gene Panels.This suggests an alteration in cellular metabolism; heat maps showed an increase in glycolytic and PPS gene transcripts in Shp2 KO rods but decreased TCA gene panels in Shp2 KO rods.Mitochondrial gene panels showed separation between rod Shp2 WT and KO mice but no discernable trend in overall transcripts, suggesting more stringent functional analysis of rod Shp2 KO rod mitochondria is needed to identify any potential functional changes.

DISCUSSION
In our current study, we meticulously characterized the cellspecific expression of Shp2 using an unbiased TRAP technique.We identified the highest enrichment of Shp2 in photoreceptor cells compared to many other retinal cell types, including Müller cells.
Additionally, staining of isolated bovine rod photoreceptors for Shp2 confirmed this observation.Shp2 is crucial for early optic cup patterning at embryonic day 9.0 (E9.0) [21].However, Six3-Cremediated ablation of Shp2 after E10.5 does not affect retinal development [21].Despite this, Six3-Cre; Shp2 fox/flox mutants exhibit ocular defects at weaning (P21), including optic nerve thinning, a dystrophic optic stalk, vacuoles, reduced retinal thickness, and rosettes in the outer nuclear layer [7].Increased apoptosis occurs in various retinal layers from P10 to P21 [7].Shp2 knockout mice generated in this study under the control of the rhodopsin promoter, which is postnatally at day 5 [22], did not exhibit retinal abnormalities or rosette formation at 6 weeks.However, by 20 weeks, they exhibited age-related photoreceptor degeneration and functional loss, indicating Shp2's cell-autonomous role in rod photoreceptors.
The tight-junction protein OCLN, identified as a substrate of Shp2 [11], is known to be expressed in the RPE [23] and observed in the outer limiting membrane [24] within the retina.Our biochemical characterization and TRAP analysis showed that OCLN expression is present in RPE, rods, and cones, but not in Müller cells.We observed a reduction in OCLN expression in the retina of rod Shp2 −/− mouse, and there was also a loss of this protein in the RPE.It has been shown that OCLN phosphorylation regulates its assembly into the tight junctions [25].In the intact epithelium, OCLN exhibits high phosphorylation on serine and threonine residues, while tyrosine phosphorylation is minimal [25].However, when tight junctions are disrupted, OCLN serine/threonine residues undergo dephosphorylation, and there is an increase in tyrosine phosphorylation in OCLN [25].In line with previous research, an in vitro study showed that when OCLN is phosphorylated at tyrosine, it no longer interacts with ZO-1, another junctional protein [25].These findings suggest that the equilibrium between tyrosine kinases and phosphatases plays a crucial role in determining OCLN phosphorylation and the function of tight junctions.Given that Shp2 is a tyrosine phosphatase, the observed reduction of OCLN in the retina and RPE of rod Shp2 −/− mice may indicate that OCLN could persist in a state of tyrosine phosphorylation, potentially causing an increased turnover.Additional comprehensive studies are necessary to confirm this hypothesis.
The TRAP/RNA sequencing highlights that Shp2 regulates several important signaling pathways in rod photoreceptor cells.The pathway analysis also identified the likely activation of upstream regulators, MLX interacting protein-like (Mlxipl), and serine/threonine kinase 11 (Stk11), and the likely inhibition of upstream regulators COP9 Signalosome Subunit 5 (Cops5), and protein tyrosine phosphatase receptor type R (Ptprr).Our study indicates that the majority of pathways regulated by Shp2 are associated with the Cell Cycle.Previous studies have identified a novel connection between Shp2 and cell cycle checkpoints [26].Additionally, Shp2 has been found to enhance the proliferation of breast cancer cells by regulating Cyclin D1 stability through the PI3K/Akt and GSK3β pathway [27].Moreover, the G2/M checkpoint induced by DNA damage in SV40 large T antigen-immortalized embryonic fibroblast cells is dependent on Shp2 [28].It is noteworthy that photoreceptor cells are postmitotic, and further investigations are required to comprehend the involvement of Shp2-regulated cell cycle regulatory pathways in photoreceptor functions.
Carbohydrate-responsive element-binding protein (ChREBP), also known as MLX-interacting protein-like (MLXIPL), is a key transcriptional regulator [29] activated by glucose, independent of insulin [30].In adipose tissue, ChREBP promotes de novo lipogenesis from glucose [31], while in the liver, it induces glycolysis and lipogenesis [30].Our study indicates that the loss of Shp2 in rod cells impacts glycolysis, the pentose phosphate pathway, the TCA cycle, and mitochondrial metabolism.In line with these findings, we also noted changes in the levels of the Glut1, glucose sensor ALDOC, hexokinase 2, and PDH in the retinas of rod Shp2 −/− mice.
In rod Shp2 −/− mice, increased GFAP expression indicates Müller cell activation.This activation, known as gliosis, occurs during ocular inflammation and involves interactions with immune and microglial cells [32].We also observed elevated inflammatory markers, but our rod photoreceptor-TRAP data showed that these markers (IL-8, IL-10, and IL-1beta) were not expressed in photoreceptor cells, suggesting they originate from nonphotoreceptor cells, possibly Müller cells.Further studies are needed to understand how Shp2 regulates retinal inflammation.
The retinal degeneration observed in rod Shp2 −/− mice may be due to reduced levels of Glut1 and increased levels of cGMP.Glut1 facilitates glucose transport from the RPE to photoreceptor cells [33], and reduced glucose supply contributes to photoreceptor cell death in inherited and age-related retinal diseases [33].Specifically, the deletion of Glut1 in rod photoreceptors restricts glucose transport to the outer retina, leading to photoreceptor degeneration and impaired outer segment renewal [33].Further studies are needed to determine if Shp2 dephosphorylates Glut1 to maintain its stability and function.Increased levels of cGMP have been shown to induce photoreceptor degeneration [34] and our metabolic analysis shows increased levels of cGMP in mouse rods lacking Shp2.Further studies are needed to examine how loss of Shp2 increases the levels of cGMP.Our study emphasizes the crucial role of Shp2 in photoreceptor cell functions, with potential phosphatase-independent functions [35] that require further investigation.

LIMITATIONS OF THIS STUDY
We observed decreased Occludin (OCLN) levels in the retina and reduced expression in the RPE, but could not assess the interaction between OCLN and Shp2 on photoreceptor function due to the lack of floxed OCLN mice.Although sex-specific metabolomics differences in mouse eyes have been reported [36], our current metabolomics data is not separated by sex.We plan to address this in future studies with a larger sample size.

Animals
All animals were treated following the ARVO Statement for using Animals in Ophthalmic and Vision Research and the NIH Guide for Care and Use of Laboratory Animals.The protocols were approved by the IACUC at the University of Oklahoma Health Sciences Center.Breeding pairs of PTPN11 (Jax # 025758), RiboTag (Jax #011029), and tamoxifen-inducible RaxCre ER mice (Jax # 025521) were purchased from The Jackson Laboratory (Bar Harbor, Maine).The rhodopsin-Cre (i75Cre) mice have been described earlier [37] and were provided by Dr. Ching-Kang Jason Chen (Baylor College of Medicine, Houston, TX).Tetracycline-inducible VMD2-Cre mice and human red/green cone opsin promoter-Cre mice were kindly provided by Dr. Yun Le (OUHSC).Animals were born and raised in our vivarium and kept under dim cyclic light (40-60 lux, 12 h light/dark cycle).All mice were screened for rd1 and rd8 mutations and were negative for these mutations.The mice were deeply anesthetized, and the retinas were harvested.The mice were euthanized by CO 2 asphyxiation.The retinas were used for RNA and protein isolation, and enucleated eyes were used for immunohistochemistry.To eliminate bias, we randomly assigned mice of the same sex, age, and genetic strain to each experimental group.We mixed litters to prevent litter bias.After genotyping and assigning unique eartag identifiers, Dr. Rajala, the principal investigator, Cell Death and Disease (2024) 15:577 randomly selected the cohorts.This ensured that the research personnel conducting the experiments were blinded and knew the mice by their eartag numbers.

Generation of conditional knockout and retinal cell-specific RiboTag mice
We have generated rod photoreceptor-specific PTPN11 (Shp2) knockout mice by breeding floxed Shp2 mice with mice carrying Cre-recombinase under the control of a rhodopsin promoter.For the identification of genotypes, we used the following primers.Rhodopsin-Cre (forward primer: TCAGTGCCTGGAGTTGCGCTGTGG and reverse primer: CTTAAAGGC-CAGGGCCTGCTTGGC) and Shp2 floxed (forward primer: ATGACTCCT-GAAGCCCATTG and reverse primer CTTCCCATCACCTCAGCATCC).The rhodopsin-Cre will identify 500 bp product.The Shp2 genotyping will generate mutant 320 bp; heterozygous 221 and 320 bp and wild type 221 bp, respectively.The RiboTag mouse carries a ribosomal protein gene (Rpl22) with a floxed C-terminal exon followed by an identical exon tagged with hemagglutinin (HA) epitope [38].We bred RiboTag mice with mice carrying Cre-recombinase under the control of rod (rhodopsin), cone (red/ green opsin), RPE (VMD2), Müller cells (Rax), and ganglion cell (CMV packaged into an AAV vector, Vector Biolabs)-specific promoters.The isolation of polyribosomes containing actively translating mRNAs and qRT-PCR analysis was carried out as described [9].

Statistical Analysis
We determined the sample size through power analysis [39].Data underwent comprehensive statistical evaluation using GraphPad Prism 7.3 software.No samples were excluded from the analysis.Various statistical methods were employed based on the experiment type.Before analysis, we conducted normality tests (Anderson-Darling, D'Agostino & Pearson, Shapiro-Wilk, Kolmogorov-Smirnov) to assess data distribution (normal or non-normal).If non-normally distributed, an unpaired non-parametric test was applied for group comparisons, involving multiple Mann-Whitney U tests.To address multiple comparisons, a false discovery rate was controlled at Q = 1%, implementing the two-stage Benjamini-Krieger-Yekutieli method.For normally distributed data, parametric tests were conducted using Welch's correction to assess significance between the two groups.The resulting p-values were used for inference.One-way ANOVA determined statistical differences among means of three or more independent groups, while a two-way ANOVA assessed how a quantitative variable's mean changes with two categorical variables.

DATA AVAILABILITY
data generated or analyzed during this study are included in this published article.The TRAP data reported here have been deposited in the Gene Expression Omnibus with accession number GSE269514.The metabolomics LC/MS Mass Spectrometry data has been deposited in the MassIVE Repository under Reference number MSV000094985.

Fig. 5
Fig. 5 Loss of Shp2 in rods alters cellular metabolism.Unsupervised hierarchical clustering glycolytic transcripts between rod fractions of rod Shp2 WT and KO mice (A).Unsupervised hierarchical clustering pentose phosphate shunt (PPS) transcripts between rod fractions of rod Shp2 WT and KO mice (B).Unsupervised hierarchical clustering TCA Cycle transcripts between rod fractions of rod Shp2 WT and KO mice (C).Unsupervised hierarchical clustering of mitochondrial transcripts between rod fractions of rod Shp2 WT and KO mice (D).Unsupervised hierarchical clustering of fatty acid oxidation transcripts between rod fractions of rod Shp2 WT and KO mice (E).Unsupervised hierarchical clustering of antioxidant transcripts between rod fractions of rod Shp2 WT and KO mice (F).

Fig. 7
Fig. 7 Changes in steady-state level retinal metabolites in mouse retina lacking Shp2.Steady-state levels of retinal metabolites were measured by LC/MS from four-independent Shp2 flox/flox (WT) and rod Shp2 −/− (KO) mouse retinas.A heatmap of changed metabolites in rod Shp2 −/− mouse retina (A).Volcano plot showing differential metabolites in rod Shp2 −/− compared to Shp2 flox/flox mouse retina, horizontal line indicates changes with (p < 0.05) (B).Quantification of TUNEL staining in rod Shp2 −/− mouse retinas.Prefer-fixed retinal sections of Shp2 flox/flox and rod Shp2 −/− mice were examined for cell death with in-situ localization of apoptosis using TUNEL and counted the number of TUNELpositive cells over the entire retina (C).Data are mean ± SEM, (n = 4).